Daniel:Notebook/RNAFISH/2014-8-30: Difference between revisions
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==Oligo Set Preparations== | ==Oligo Set Preparations== | ||
These are the oligos that I ordered on | These are the oligos that I ordered on [[Daniel:Notebook/RNAFISH/2014-8-4|08/04/2014]]. Now that I am ready to do the experiment, I need to prep them. First thing is to resuspend the oligos. By order, each oligo has 15 nmol. | ||
#Resuspend each oligo in 15 uL nfH2O (1 mM) | |||
#Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM) | |||
#Add 432 uL nfH2O, diluting 10:1, for final concentration of 100 uM | |||
#Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy | |||
===Nanodrop Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B7DEE8;font-size:12pt" align="center" | |||
| width="65" height="33" | Sample | |||
| width="65" | 1:1 Measured | |||
| width="65" | 5:1 Measured | |||
| width="65" | 5:1 Corrected | |||
| width="65" | 10:1 Measured | |||
| width="65" | 10:1 Corrected | |||
|style="font-weight:bold" width="65" | Average | |||
|style="font-weight:bold" width="65" | Standard Deviation | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | GAD1 | |||
| align="center" align="center" | 558 | |||
| align="center" align="center" | 109.3 | |||
| align="center" align="center" | 546.5 | |||
| align="center" align="center" | 43.2 | |||
| align="center" align="center" | 432 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 512.2 | |||
| align="center" align="center" | 69.7 | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | FOXP2 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 554.3 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 114.9 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 574.5 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 54.5 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 545 | |||
|style="background-color:#D9D9D9;font-weight:bold" align="center" align="center" valign="bottom" | 557.9 | |||
| align="center" align="center" | 15.1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RBFOX3 | |||
| align="center" align="center" | 558.9 | |||
| align="center" align="center" | 115.2 | |||
| align="center" align="center" | 576 | |||
| align="center" align="center" | 56.8 | |||
| align="center" align="center" | 568 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 567.6 | |||
| align="center" align="center" | 8.6 | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | CUX2 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 582.2 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 124.9 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 624.5 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 60.6 | |||
|style="background-color:#D9D9D9" align="center" align="center" | 606 | |||
|style="background-color:#D9D9D9;font-weight:bold" align="center" align="center" valign="bottom" | 604.2 | |||
| align="center" align="center" | 21.2 | |||
|} | |||
So each one has around 0.5 ug/uL. Pretty good. |
Revision as of 23:15, 30 August 2014
Trial Run (Started 08/29/2014)
Tissue Section Preparation (From 08/29/2014)
- Wash the tissue in PBS, 3 × 30 min
- Incubate the tissue in a sucrose series with increasing concentrations:
- 10% for 1 h
- 20% for 1 h
- 30% at 4C overnight
Oligo Set Preparations
These are the oligos that I ordered on 08/04/2014. Now that I am ready to do the experiment, I need to prep them. First thing is to resuspend the oligos. By order, each oligo has 15 nmol.
- Resuspend each oligo in 15 uL nfH2O (1 mM)
- Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
- Add 432 uL nfH2O, diluting 10:1, for final concentration of 100 uM
- Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
Nanodrop Results
Sample | 1:1 Measured | 5:1 Measured | 5:1 Corrected | 10:1 Measured | 10:1 Corrected | Average | Standard Deviation |
GAD1 | 558 | 109.3 | 546.5 | 43.2 | 432 | 512.2 | 69.7 |
FOXP2 | 554.3 | 114.9 | 574.5 | 54.5 | 545 | 557.9 | 15.1 |
RBFOX3 | 558.9 | 115.2 | 576 | 56.8 | 568 | 567.6 | 8.6 |
CUX2 | 582.2 | 124.9 | 624.5 | 60.6 | 606 | 604.2 | 21.2 |
So each one has around 0.5 ug/uL. Pretty good.